We have recently shown that apolipoproteins (apo) E3 and E4 have differential effects on neurite outgrowth from dorsal root ganglion neurons in vitro, suggesting that apoE could play a direct role in modulating neuron development or remodeling. Only apoE added to the cell with beta- migrating very low density lipoproteins (beta-VLDL) or lipid emulsions had an effect, and the effect was blocked by reductive methylation of apoE or by antibodies to the receptor-binding domain of apoE. We have further demonstrated that microtubule stability is altered in apoE4-treated cells. The major goals of this proposal are 1) to determine the lipoprotein and lipid requirements mediating the differential effects of apoE3 and apoE4 on neurite outgrowth; 2) to determine if the low density lipoprotein (LDL) receptor, heparan sulfate proteoglycans (HSPG) and/or the LDL receptor- related protein (LRP) are required for the differential effects; 3) to examine in detail the binding, internalization, and degradation of apoE3 and apoE4 by neurons; and 4) to determine the mechanism by which apoE3 and apoE4 exert differential effects on neurite outgrowth. To accomplish these goals, in vitro studies will be performed with primary cultures of neurons, and a murine neuroblastoma cell line, Neuro-2a. The lipid and lipoprotein requirements for the effect will be determined by incubating the cells with apoE3 or apoE4 together with the naturally occurring lipoproteins LDL or apoE HDLc or with lipid emulsions containing different ratios of cholesterol, cholesteryl ester, triacyglycerol, and phospholipid, and determining the effects on neurite outgrowth. The importance of the LDL receptor, LRP, and HSPG to the differential effects of the apoE isoforms will be explored using: neurons from LDL receptor knockout animals; competition studies with lactoferrin and the 39-kDa protein; and mutants of apoE (apoE2, apoE(Arg142 yieldsCys), apoE Leiden) with various degrees of defective binding to the LDL receptor, the LRP, and HSPG. The effect of the apoE variants will be examined both when they are added exogenously together with beta-VLDL and when beta-VLDL is added to cells stably expressing the apoE variants. Internalization and degradation of the apoE3 and apoE4 with beta-VLDL will be examined in detail to determine the reason for our observation that apoE3 and apoE4 with beta-VLDL will be examined in detail to determine the reason for our observation that apoE3 accumulates in the cells to a greater extent than apoE4. In addition, light-level and electron microscopy will be used to determine the location of intracellular apoE and its association with organelles of importance in neurite outgrowth. In the final Specific Aim we will determine if truncated apoE can mediate the differential effects, and we will determine the mechanism by which apoE affects microtubule formation. These studies will provide an understanding of the mechanisms that account for the differential effects of apoE3 and apoE4 on neurite outgrowth in vitro and potentially will explain the reason for the association of the apoE4 allele with Alzheimer's disease.